1. Field of the Invention
This invention relates to the field of recombinant technology of viruses and to production of recombinant viral vectors, and is particularly related to production of recombinant adenoviral vectors with reduced levels of contamination by replication competent adenoviruses (RCA).
2. Background of the Invention
As taught in WO95/00655, adenoviruses (Ads) in the form of recombinant adenoviral vectors can be used as mammalian cell expression vectors, with excellent potential as live recombinant viral vaccines, as transducing vectors for gene therapy, for research, and for production of proteins in mammalian cells (see Hitt, M., Addison, C. and Graham, F. L Human adenovirus vectors for gene transfer into mammalian cells. In: “Advances in Pharmacology—Gene Therapy” Ed. J. Thomas August, Academic Press. San Diego, Calif. 40: 137–206, 1997 for review). General uses for recombinant adenoviral vectors also include use in functional genomics research, protein over-expression, pre-clinical studies and clinical trials.
In the human Ad genome, early region 1 (E1), E3, and a site upstream of E4 have been utilized as sites for introducing foreign DNA sequences to generate adenovirus recombinants. In the absence of compensating deletions in E1 or E3, a maximum of about 2 kb can be inserted into the Ad genome to generate viable virus progeny (Bett, A. J., Prevec, L., and Graham, F. L. Packaging capacity and stability of human adenovirus type 5 vectors. J. Virol. 67: 5911–5921, 1993.). The E1 region is not required for viral replication in complementing 293 cells (Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–72, 1977.), or other cells known to complement E1, and up to approximately 3.2 kb can be deleted in this region to generate vectors with a capacity of 5.0–5.2 kb. Besides expanding the capacity of the vector to permit insertion of larger amounts of foreign DNA, deletion of E1 results in a defective Ad which is essentially unable to replicate in normal cells and thus provides a measure of safety as well as increased utility for many applications employing these vectors. In the E3 region, which is not required for viral replication in cultured cells, deletions of various sizes have been utilized to generate nonconditional helper independent vectors with a capacity of up to 4.5–4.7 kb. The combination of deletions in E1 and E3 permits the construction and propagation of adenovirus vectors with a capacity for insertions of up to approximately 8 kb of foreign DNA.
Recombinant adenoviral vectors with foreign DNA inserted in place of E1 sequences, and optionally also carrying deletions of E3 sequences, are conventionally known as “first generation” (FG) recombinant adenoviral vectors. FG vectors are of proven utility for many applications. They can be used as research tools for high-efficiency transfer and expression of foreign genes in mammalian cells derived from many tissues and from many species. First generation vectors can be used in development of recombinant viral vaccines when the vectors contain and express antigens derived from pathogenic organisms. Importantly, Recombinant adenoviral vectors have become extensively used for human gene therapy, because of their ability to efficiently transfer and express foreign genes in vivo, and due to their ability to transduce both replicating and nonreplicating cells in many different tissues. Adenovirus vectors are widely used in these applications, as well as other applications disclosed herein and in the references cited herein. The biology and applications for adenoviruses is described in Adenoviral Vectors for Gene Therapy, David T. Curiel and Joanne T. Douglas, Academic Press, 2002 (and in particular Chapters 3, 4, 5, 6, 15 and 16).
The construction of recombinant adenoviral vectors can be performed in many ways (reviewed by Ng, P. and Graham, F. L. Construction of first generation adenoviral vectors. In: Methods in Molecular Medicine. Gene Therapy Protocols, 2nd edition. Jeffrey R. Morgan (Ed). Humana Press Inc. Totawa, N.J. Vol. 69, pp. 389–414, 2002 and by Hitt, M., Bett, A. J., Prevec, L. and Graham, F. L Construction and propagation of human adenovirus vectors. In: “Cell Biology: A Laboratory Handbook” Ed. J. E. Celis. Academic Press. 2nd Edition, Vol. 1, pp 500–512, 1998.) The most popular methods for isolation make use of recombination between a small shuttle plasmid containing sequences from the left end of the viral genome and typically comprising an insert of foreign DNA and a genomic plasmid that comprises the remainder of the viral genome. Recombination in cotransfected E1+ host cells such as 293 cells results in formation of the desired FG vector. (See Bett, A. J., Haddara, W., Prevec, L. and Graham, F. L An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. US 91: 8802–8806, 1994. and U.S. Pat. No. 6,140,087 for details). An improvement in this system was made by utilizing site specific recombinases such as Cre or FLP acting on loxP and frt target sites respectively to induce recombination between the shuttle plasmid and the genomic plasmid. Example of such systems are described in Ng, P., Parks, R. J., Cummings, D. T., Evelegh, C. M., Sankar, U. and Graham, F. L. An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method. Human Gene Therapy 11: 693–699, 2000; Ng, P., Cummings, D. T., Evelegh, C. M. and Graham, F. L. The yeast recombinase FLP functions effectively in human cells for construction of adenovirus vectors. BioTechniques 29: 524–528, 2000; Ng, P. and Graham, F. L. Adenoviral Vector Construction I: Mammalian Systems In: Adenoviral Vectors for Gene Therapy. D. T. Curiel & J. T. Douglas (Eds) Academic Press, NY. 2002, pp 71–104. Site-specific recombination catalyzed by an efficient recombinase, such as the Cre or FLP recombinase, can be many-fold more efficient than homologous recombination. This methodology is also applicable to insertion of foreign DNA sequences into various regions of the viral DNA in addition to, or instead of, the E1 region classically used for that purpose.
Another popular method for construction of Recombinant adenoviral vectors makes use of homologous recombination in bacteria to substitute viral DNA sequences with foreign DNA (C. Chartier, Degryse, E; Gantzer, M; Dieterle, A; Pavirani, A; Mehtali, M Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J. Virol. 70 (1996), pp. 4805–4810.). The use of homologous recombination in bacteria is also applicable to insertion of foreign DNA sequences into various regions of the viral DNA, in addition to the E1.
A typical first generation recombinant adenoviral vector is illustrated in FIG. 1. It has a deletion of the E1A and E1B gene sequences of the Early Region I and a substitution of those E1 sequences with foreign DNA usually comprising a promoter, a cDNA, and a polyadenylation signal together comprising an expression cassette. Because E1 encoded functions are necessary for replication of adenoviruses FG vectors must be propagated in mammalian cells that contain and express E1 functions. The cell line most commonly used for this purpose is the 293 cell line (Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–72, 1977.) which contains adenovirus type 5 (“Ad5”) sequences extending from nucleotide (“nt”) 1 to nt 4344 integrated into chromosome 19 (19q13.2) (Louis, N., Evelegh, C. and Graham, F. L. Cloning and sequencing of the cellular/viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology, 233:423–429, 1997.) The deletion of E1 sequences from FG vectors usually does not encompass the ITR from nt 1 to about nt 103 nor the viral DNA packaging signal from about nt 190 to about 380 nor the coding sequences for protein IX because the ITR and packaging signal are required in cis for viral DNA replication and packaging of the vector DNA into virion capsids, respectively, and the pIX protein is a required capsid component of the virion.
The requirement for pIX coding sequences is interesting for a number of reasons. Firstly the protein was originally thought to be dispensable for virus production because viruses with deletions of E1 that included the pIX gene were viable (though the virions produced were more heat labile than wt virions) (Colby, W. W., and Shenk, T. (1981). Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX. J. Virol. 39, 977–980.). However it was subsequently shown that pIX is essential for efficient packaging of full length viral genomes into functional virions (Ghosh-Choudhury, G., Haj-Ahmad, Y., and Graham, F. L. (1987), Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes. EMBO J. 6, 1733–1739, Sargent, K. L., Ng., P., Graham, F. L. and Parks, R. J. Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors. Gene Ther. 11: 504–511, 2004.) Although 293 cells contain the pIX coding sequences the gene is not expressed in 293 cells, hence the need to include the pIX gene in the Ad vector genome for virion stability and for efficient packaging of full length virion DNA.
A major problem in the production of FG recombinant adenoviral vectors in 293 cells and many other E1 complementing host cells is the appearance of replication competent adenoviruses (RCA) as contaminants of the vector stock (Lochmuller, H., Jani, A., Huard, J., Prescott, S., Simoneau, M., Massie, B., Karpati, G., and Acsadi, G. (1994): Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (ΔE1-ΔE3) during multiple passages in 293 cells. Hum. Gene Ther. 5, 1485–1492.) RCA appear as a result of recombination between vector DNA sequences and homologous sequences in the host cells as discussed below. For many applications the presence of RCA in vector stocks is a significant problem (discussed by Fallaux, F. J., van der Eb, A. J. and Hoeben, R. C. Who's afraid of replication-competent adenoviruses? Gene Ther. 6: 709–712, 1999) and numerous attempts have been made to solve this problem by modifying the vectors, the complementing host cells, or both.
The structure of Ad5 sequences integrated in the 293 cellular genome is illustrated in FIG. 1. As mentioned above, the viral sequences comprise an uninterrupted segment of Ad5 extending from the first nt of the Ad5 genome to nt 4344 and contain therefore, an intact left ITR, the complete packaging signal and a complete E1 region composed of E1A and E1B. Also contained in the cell are the coding sequences for pIX and part of the leftward-transcribed IVa2 gene. Indicated immediately above the Ad5 sequences is a typical E1 deletion used in FG vectors to eliminate E1 and to allow for substitution of foreign DNA. Numerous similar FG vectors have been constructed and described in which the end points of the deletions may vary but generally the deletions cannot extend significantly further left or right of those end points shown in FIG. 1 without compromising the ability of the vector to replicate. It can be seen that there is significant overlap of homologous sequences between the resulting FG vector DNA and the Ad5 DNA of 293 cells on either side of the deletion. Without being bound to a particular theory, recombination event(s) along these regions of homologous sequences is/are believed to be the reason for occurrence of RCA. Because 293 cells contain an intact left ITR as well as the packaging signal it is theoretically possible to generate an E1+virus by a single recombination event involving the sequences to the right of the E1 deletion, thus joining the vector DNA to the E1 sequences of 293 cells. However, Hehir et al. have demonstrated that RCA can occur, and indeed more frequently (perhaps always) do occur as a result of two recombination events, one on each side of the E1 region (Hehir, K. M., Armentano, D., Cardoza, L. M., Choquette, T. L., Berthelette, P. B., White, G. A., Couture, L. A., Everton, M. B., Keegan, J., Martin, J. M., Pratt, D. A., Smith, M. P., Smith, A. E. and Wadsworth, S. C. (1996). Molecular characterization of replication-competent variants of adenovirus vectors and genomic modifications to prevent their occurrence. J. Virol. 70,8459–8467.). This implies that the presence of an intact ITR in the complementing cells is not necessary for generation of RCA provided sufficient overlap is present on each side of the E1 deletion.
Hehir et al. attempted to reduce the frequency of RCA generation during growth of Ad vectors in 293 cells by modifying the design of their vectors. In one strategy they deleted or rearranged the coding sequences for the pIX protein that are at the 3′ end of E1 and effectively extended the E1 deletion to nt 4020. This strategy was followed to reduce the degree of overlap between Ad sequences in 293 cells and the sequences to the right of E1 in the vector and thereby, per their theory, reduce the efficiency of recombination and the consequent formation of RCA. In fact, as was subsequently shown in the inventor's laboratory, the Ad5 sequences integrated in the genome of 293 cells extend considerably beyond the pIX gene to nt 4344 implying overlap to the extent of about 320 bp. Consequently removal of the pIX gene would not be expected to prevent absolutely the occurrence of RCA. Hehir et al. claimed that although RCA contamination was reduced, it was not absolutely eliminated.
Other approaches to reduce the frequency of RCA contamination have involved modifications not to the vectors but to the viral DNA sequences used to establish E1+complementing host cells. Thus, for example, Imler et al. (Imler, J. L., Chartier, C., Dieterle, A., Sainte-Marie, M., Faure, T., Pavirani, A. and Metali, M. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors. Gene Ther. 3: 75–84, 1996) developed complementing cell lines that lacked the left ITR and packaging signal of the Ad5 genome and in which E1A expression was regulated by heterologous promoters. They included the pIX gene in the plasmids used to transform A549 cells but observed that pIX was not efficiently expressed in any of the transformed clones as is also the case in 293 cells. Their vectors retained the left ITR and the packaging signal and had an insertion of foreign DNA substituting for E1 coding sequences. The vectors had a deletion of E1 sequences extending from nt 459 to at least Ad5 nt 3510 whereas the complementing cells had Ad sequences extending from nt 505 to nt 3510. Consequently the vectors and cell lines had no homology at the extreme left end or right end of E1, i.e., to the left or right of the foreign DNA insert. This was predicted to prevent or reduce the efficiency of RCA generation though they did not present any experimental data bearing on this. Unfortunately the host cells only complemented E1 deleted vectors about 1/10 as well as did 293 cells. A somewhat similar strategy was described by Brough and Kovesdi (U.S. patent application 2003/0040100) who established A549 cells transformed with an expression cassette comprising adenovirus type 2 (“Ad2”) sequences from nts 362 to 5708 with the E1A promoter enhancer replaced by the HCMV IE gene promoter. Again, the cells were expected to support replication of an Ad vector (with an E1 deletion of nts 356 to 3328) without generation of RCA by virtue of the fact that there is no overlap left of E1 and consequently no possibility of homologous recombination events capable of generating RCA. Another approach was adopted by Fallaux et al (Fallaux, F. J., Bout, A., van der Velde, I., van der Wollenberg, D. J., Hehir, K. M., Keegan, J., Auger, C., Cramer, S. J., van Ormondt, H., van der Eb, A. J., Valerio, D., and Hoeben, R. C. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum. Gene Ther. 9: 1909–1917, 1998, and see also U.S. Pat. Nos. 6,670,188; 6,692,966; 6,602,706; 6,306,652; 6,265,212). Fallaux et al. developed cell lines transformed by plasmids comprising Ad5 nts 459–3510 expressed under the control of the human phosphoglycerate kinase (PGK) promoter and used the resulting complementing E1+cells, typified by the PER.C6 line, for growth of “matched” E1 deleted Ad vectors that had no homology whatsoever with the viral sequences in PER.C6 cells, either to left or to right of the E1 deletion. They reported that matching the host cells and the vectors in this way solved the RCA problem. Unfortunately, unlike 293 cells, PER.C6 cells are not readily available and are expensive to obtain. Consequently it would be most valuable if alternate approaches could be developed, especially systems that permit the use of 293 cells. See for example Davidson et al (U.S. patent application 2002/0098571, page 3, paragraph 0023) who pointed out “This (ability to use standard 293 cells) is important since most investigators do not have access to alternative cell lines, or cannot justify the cost of their use.”
The investigators whose work is outlined above did not consider modifying the packaging signal of the vector as a possible approach to solving the RCA problem but instead focused on modifying the left end viral DNA sequences in the complementing host cells, essentially eliminating the ITR and packaging signal (which overlaps with the E1A enhancer promoter) and substituting these viral control elements with a heterologous promoter such as the PGK promoter or the HCMV IE promoter. One group, however, has made certain recombinant virus constructs comprising an inverted packaging signal (Palmer, D and Ng, P. Improved system for helper-dependent adenoviral vector production. Molec. Ther. 8: 846–852, 2003). They constructed a helper virus for amplification of helper dependent vectors in the system described in U.S. Pat. No. 5,919,676 wherein the packaging signal of the helper was inverted with respect to the packaging signal of the helper dependent vector. The purpose of this modification was to avoid recombination between the left end sequences of the helper dependent vector and helper that would effectively remove a loxP site from the helper virus DNA and render it resistant to the effects of Cre recombinase whose action is otherwise needed to excise the packaging signal of the helper virus to prevent packaging of helper virus DNA into virions. Palmer and Ng also made another modification in the helper, inserting a non-coding DNA stuffer into the E3 region. The stated purpose of adding the stuffer sequence was to render any recombinants comprising this part of the helper DNA too large to package into RCA. (See Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. and Graham, F. L. A new helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. U.S. 93: 13565–13570, 1996.)
Despite varied and dedicated research efforts by a number of research groups to effectively and efficiently reduce RCA generation in the propagation of first generation recombinant adenoviral vectors, there exists a need to identify and utilize systems, methods and compositions that provide for the reduction or elimination of RCA during such propagation. The present invention addresses this need.